Globally disseminated Shiga toxin (Stx)-producing Escherichia coli (STEC) are notorious for producing a phage-borne cytotoxin, that is direct mediator of lethal food borne disease. Human morbidity and mortality remain unacceptably high, as no vaccines and only a limited arsenal of therapeutic or preventive countermeasures are available. Progression to life-threatening complications during human infection, such as HUS, is inexorably linked to the production of the most potent cytopathic toxin subtype Stx2a. Hypervirulence as manifested by increased Stx2a titers has been associated with circulating subpopulations through phylogenetic, epidemiological and phenotypic linkage. However, there is a dearth of knowledge of the intrinsic genomic make-up of high-level Stx2a producers. The research objective of this proposal is to apply a systematic and genome-scale approach to identify causal pathogenome loci responsible for hypervirulent toxin production in the STEC pathogenome. The central hypothesis is that differences in the isolates' individual Stx2a production capability are correlated with strain-level sequence variation anchored in both the carried Stx2a-Phage Sequence Type (PST) and external loci on the phage-hosting STEC pathogenome. The three specific aims to test this hypothesis are as follows: 1) To catalogue genome-scale variation in clinical STEC specimen through holistic Whole Genome Sequencing Typing of the STEC core and accessory Stx2a-phage inventory. 2) To characterize a culture bank of Stx2a lysogen. To reduce the genomic complexity we will create a genomically defined variant of Stx2a-lysogen cultures. Through lysogenic conversion we will introduce archetypical Stx2a- PST into the genome background of non-shigatoxigenic E. coli hosts, using among others, atypical stx- negative STEC and resident gastrointestinal E. coli from our culture collection. Engineered Stx2a-lysogens will provide a controlled genomic testbed to systematically determine how the Stx2a-PST, phage dosage and chromosomal location impart Stx2a production, independently or in combination with loci external to the phage on the respective host chromosomes, and 3) To identify causal genome loci responsible for hypervirulent Stx2a production. The synergistic determination of phylogroup and Stx2a-production pathotype in wt STEC and engineered Stx2a-lysogens provides a robust foundation to identify modulatory loci of Stx2a production through Genome Wide Association Studies. Cultures are phenotyped for Stx2a-phage mobilization efficiency, global transcriptome changes, Stx2a titers, and in vivo cytotoxicity. Each genotypic variable is tested individually and in groups of genotypes to account for the participation and interplay of polymorphic Stx2a phage and/or host loci. This research will provide attractive targets for the development of improved biosurveillance, risk assessment of suppressive therapeutic anti-Stx2a strategies. Due to the evolutionary conservation among Stx2a-phages and phage-hosting STEC pathogenomes, we anticipate translational application of developed principles and identified Stx2a regulatory loci for the collective group of clinically important priority STEC.